(from abstract). Recurrent herpes simplex virus type 1 (HSV-1) infection is a major cause of viral induced blindness. LAT, the only HSV-1 gene previously thought active during latency, is important for efficient reactivation of HSV-1. The PI has recently discovered a novel HSV-1 gene (AL) that, like LAT, is also transcriptionally active during latency, and is therefore a candidate for involvement in the latency-reactivation cycle. AL is antisense to LAT and overlaps the LAT promoter and the 5' end of LAT. The AL-RNA is polyadenylated and contains an ORF. Deletion of most of the AL-ORF and the corresponding region of LAT, produced a mutant with increased virulence and decreased spontaneous reactivation, suggesting that (1) AL and/or LAT are involved in spontaneous reactivation, and (2) AL-protein plays a role in virulence.
The specific aims i nclude: 1. Fine map and characterize the AL gene, RNA, and protein. Mapping will employ RT-PCR, RPA primer extension, RACE and cDNA cloning and sequencing, of HSV-1 RNA from tissue culture and rabbit trigeminal ganglia. Anti-AL protein antibody will be made and used to confirm the presence of an AL-protein. The time of AL expression will be determined. 2. Determine the function of AL. AL-/LAT+ mutants will be constructed and analyzed in rabbits for the effect on virulence and spontaneous reactivation. Since AL and LAT overlap, knocking out one gene without altering function of the other is complex. Approaches include: (a) single nucleotide changes in the AL-ORF ATG that knockout AL-protein without altering LAT RNA structure; (b) minimal LAT promoter alterations to block LAT transcription without altering the AL-protein amino acid sequence; and (c) expression of AL-protein from an alternative site in AL-/LAT+ and AL-/LAT- mutants. The construction and analysis of these complementary AL-/LAT+ and AL+/LAT- mutants should allow us to determine whether AL is involved in spontaneous reactivation and/or virulence.
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